Light microscopy has been an irreplaceable tool in life sciences for several centuries. Nevertheless, its design has not fundamentally changed since its inception, i.e., the image of the specimen is magnified through a system of lenses and other optical components before being detected by the eye or a digital sensor array for visualization. The quest to resolve smaller features with better resolution and contrast has improved the capabilities of light microscopy at the cost of increasing its size and complexity. On the other hand, emerging technologies have flourished such as microfluidic and lab-on-a-chip systems which offer fast and efficient handling and processing of biological samples within highly miniaturized architectures. However, optical inspection of specimens is still being performed by conventional light microscopes, which has in general several orders of magnitude size mismatch compared to the scale of the microfluidic systems. As a result, there is a clear need for alternative compact microscopy modalities that are capable of integrating with miniaturized lab-on-a-chip platforms.
The urge for new optical microscopy modalities is not solely driven by the need for miniaturization and microfluidic integration. The fact that high resolution is achieved at the cost of significant field-of-view (FOV) reduction is another fundamental limitation of lens-based imaging. The relatively small FOV of conventional light microscopy brings additional challenges for its application to several important problems such as rare cell imaging or optical phenotyping of model organisms, where high throughput microscopy is highly desired.
In order to provide a complementary solution to these aforementioned needs, alternative, lens-free microscopy platforms have been developed which combines high resolution and large FOV in a compact, on-chip imaging architecture. In this modality, digital in-line holograms of micro-objects are recorded on a sensor array using partially coherent illumination with unit fringe magnification such that the entire active area of the sensor serves as the imaging FOV. To overcome the resolution limitation imposed by the pixel size at the sensor, multiple sub-pixel shifted holograms of the sample are acquired, and pixel super-resolution techniques are then applied to achieve sub-micron lateral resolution without compromising the large FOV. As a result, a lateral imaging performance comparable to a microscope objective with a numerical aperture (NA) of ˜0.5 has been achieved over an FOV of 24 mm2, which is more than two orders-of-magnitude larger than that of an objective lens with similar resolution. See e.g., Bishara W. et al., Lensfree on-chip microscopy over a wide field-of-view using pixel super-resolution. Optics Express 18:11181-11191 (2010).
While pixel super-resolution techniques in partially coherent lens-free in-line holography enable imaging with sub-micron lateral resolution over a large FOV, the axial resolution is unfortunately significantly lower (e.g., >40-50 μm) due to the inherently long depth-of-focus of digital in-line holography. Accordingly, despite the fact that holographic reconstruction can be numerically focused at different depths, sectioning of planes closer than ˜50 μm has not been feasible with lens-free wide-field holographic microscopes regardless of their detection numerical apertures. This fundamental limitation needs to be addressed.
Along the same lines, in recent years, there has been an increased interest in optical microscopy modalities that enable sectional imaging. As an example, Optical Projection Tomography (OPT) has been proposed, where an optically cleared specimen immersed in index-matching gel is rotated with respect to the fixed optical path of a conventional lens-based microscope, offers an isotropic resolution of ˜10 μm in all three dimensions within an imaging volume of up to ˜1 cm3. See Sharpe J et al., Optical Projection Tomography as a Tool for 3D Microscopy and Gene Expression Studies, Science 296:541-545 (2002).
A modified version of OPT by using high NA objective lenses has also been implemented recently to achieve sub-micron resolution cell imaging over a significantly reduced volume of e.g., <0.0005 mm3 See Fauver M et al., Three-dimensional imaging of single isolated cell nuclei using optical projection tomography, Optics Express 13:4210-4223 (2005).
Optical Diffraction Tomography (ODT) is another powerful technique where digital holography is utilized to reconstruct the 3D refractive index distribution of the specimen by changing the illumination direction, rotating the object, or by capturing multiple images at different wavelengths. These tomographic systems can routinely image cells potentially achieving sub-micron resolution in all three dimensions. However the trade-off between resolution and imaging volume also applies to these systems just like conventional microscopy, and high resolution is achieved at the cost of a significantly reduced imaging FOV of e.g., less than 0.04-0.2 mm2 and a depth-of-field (DOF) of less than 10-20 μm depending on the objective lens that is used.
For the same purpose, another imaging modality, namely, Selective Plane Illumination Microscopy (SPIM) has also been introduced, which utilizes a light sheet generated by a cylindrical lens to successively illuminate selective planes within a fluorescent sample to create a 3D image with enhanced axial resolution. See Huisken J et al., Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy, Science 305:1007-1009 (2004). SPIM, which is limited to only fluorescent imaging, achieves ˜6 μm axial resolution in thick samples up to a few millimeters over an FOV ranging between 0.04-2 mm2, which is dictated by either the NA of the objective lens that is used or the active area of the opto-electronic sensor array. In general, these existing optical tomography platforms, as summarized above, all rely on relatively complex and bulky optical setups that are challenging to miniaturize and integrate with microfluidic systems. Therefore, an alternative tomographic microscopy platform which offers both high resolution and a large imaging volume in a compact embodiment may offer an important imaging toolset in various fields including cell and developmental biology, neuroscience and drug discovery.